Clinical analysis of 163 pediatric patients with infectious mononucleosis: a single‐center retrospective analysis

Abstract Objective This study aims to enhance the management of Epstein‐Barr Virus (EBV) infections by analyzing the correlation between laboratory indicators and clinical manifestations in children, thereby proposing more precise diagnostic and treatment strategies. Methods In this retrospective study included 163 pediatric patients with EBV infections treated at the Children's Hospital of Soochow University from December 2017 to December 2019. Data collected through retrospective analysis included gender, age, clinical symptoms, signs, liver function tests, T‐cell subset distribution, EBV‐DNA copy numbers in plasma, and treatment outcomes. Patients were grouped based on EBV‐DNA copy numbers in plasma and hospital stay duration to compare clinical indicators across different groups. Results The dichotomous results of EBV‐DNA copy numbers in plasma showed that the two groups of children were significantly different in the number of days of fever (p = .0022), platelet count (p = .0212), ALT (p = .001), immunoglobulin IgM (p = .0039), IgG (p = .0195), TBiL (p = .025), LDH (p = 0.0001), and length of hospital stay (p < .001) were significantly different, indicating that EBV‐DNA copy numbers in plasma may be correlated with these characteristic variables. The dichotomous results of the length of hospital stay showed that the two groups were significantly increased in tonsil enlargement (p = .0024), platelet count (p = .0059), LDH (p = .0394), and ferritin (p = .0106) and EBV‐DNA copy numbers in plasma (p = 0.0361) were significantly different, This suggests a potential correlation between EBV‐DNA copy numbers in plasma and these clinical indicators. Conclusion Variations in platelet counts and lactate dehydrogenase (LDH) levels in children with EBV infections may serve as indicators of clinical outcomes.

The Epstein-Barr Virus (EBV), also known as Human Herpesvirus 4, is a ubiquitous double-stranded DNA virus found in approximately 95% of the global population 1 and a significant pathogen in children. 2 In developed regions like Europe and the USA, primary infections typically occur in late adolescence. 3However, in developing regions, infections often occur earlier, 4 possibly due to differences in socioeconomic status.Improved economic conditions and higher education levels in coastal areas of China may have led to changes in the epidemiological features of EBV infections, necessitating additional research.EBV infections are common viral infections in children, presenting various symptoms. 5Primary EBV infections during childhood are often asymptomatic or atypical, 6 which could lead to misdiagnoses or missed diagnoses.
Approximately 50% of children under 6 years old with primary EBV infection present with infectious mononucleosis (IM).Epidemiological data from China indicate that the seropositive rate for EBV infection among children aged 3 to 5 years is between 80.7% and 100.0%, reaching 100.0% by the age of 10.IM is essentially a self-limiting lymphoproliferative disorder with a generally good prognosis, and a mortality rate of only 1-2%.Chronic active EBV infection (CAEBV) is a rare manifestation of EBV in children, characterized by recurrent or persistent IM-like symptoms lasting for several months.CAEBV has a poor prognosis, with a mortality rate of up to 43%, often affecting multiple systems, including the hematological, digestive, respiratory, nervous, and cardiovascular systems. 7In rare cases, EBVinfected children may develop EBV-associated hemophagocytic lymphohistiocytosis (EBV-HLH), a rapidly progressive and often fatal condition if not promptly diagnosed and treated, with a mortality rate of 30-40%.The primary EBV infection is associated with systemic interferon (IFN) responses. 8EBV-mediated activation of plasmacytoid dendritic cell (pDC) TLR 9 can lead to the secretion of IFN-α, a key factor in controlling EBV infection. 9Therefore, the innate immune response plays a crucial role in the body's defense against EBV.Infectious mononucleosis is the most common disease caused by EBV infection in children.
Moreover, there are few large-sample studies on primary and reactivated EBV infections in children, and the mechanisms of asymptomatic EBV infections in children are not well understood. 10This study aims to further explore the clinical and laboratory characteristics of EBV infections in children to fill current research gaps.
In this study, we selected 163 children with EBV infections treated in our hospital, reviewed their clinical data, and grouped them by EBV copy numbers and hospital stay durations to compare clinical indicators across groups, thereby clarifying the correlation between laboratory and clinical indicators in children with EBV infections to improve diagnosis and treatment.

| Study subjects
We collected data from 163 children with EBV infections who were hospitalized for treatment at the Children's Hospital of Soochow University from December 2017 to December 2019.

| Inclusion criteria
1) Aged ≤18 years; 2) Met the diagnostic criteria for EBV infection; 3) Only first-time clinical data from our hospital were collected; 4) Complete clinical data available; 5) Excluded co-infections, malignant tumors, congenital malformations, severe organ dysfunction; 6) Severe complications or had received EBV treatment were excluded.

| Clinical data collectionh
This study collected the following data upon hospital admission: age, gender, clinical manifestations, complete blood count results, biochemical panel (including liver function indices), humoral immunity, cellular immune function, and final outcomes.

| Analysis
1) Statistical analysis was conducted on the gender, age, clinical manifestations, laboratory results, diagnosis, and outcomes of 163 children with EBV infections; 2) Divide the children into two groups based on EBV levels in the plasma: those with EBV-DNA copy numbers in plasma (×10 2 ) > 38.9 and those with levels ≤38.9, and compare the clinical manifestations and laboratory results between these groups.3) Further categorize the 163 children based on the number of hospital stay days into two groups: those with more than 9 days and those with 9 days or fewer, and compare the clinical and laboratory results between these groups.

| Statistical methods
Data analysis was performed using the R software package.Categorical variables were analyzed using the chi-squared test with dplyr, tidyr, and victim packages.Continuous variables were analyzed using t-tests or non-parametric tests with dplyr, reshape, and restatix packages.A p-value < .05indicated statistical significance, and p-value < .01indicated a significant difference.

| Ethics committee approval
This study was approved by Children's Hospital of Soochow University ethics committee and followed relevant ethical guidelines.Due to the retrospective nature of the study, patient consent was not required.

| Basic patient information
This study evaluated 163 pediatric patients diagnosed with EBV infections.Detailed demographic and baseline clinical characteristics are summarized in Table 1, which provides insights into the epidemiological features of EBV in this cohort.

| Clinical presentation and epidemiological insights
Fever: The majority of patients (95.71%) presented with fever, exhibiting a peak median temperature of 39.0°C.The median duration of fever was 6 days, indicative of acute infection phases in the majority of cases.

| Hematological and biochemical profiles
Complete Blood Count: The analysis included white blood cells, neutrophil percentage, lymphocyte percentage, absolute lymphocyte count, platelets, hemoglobin, and C-reactive protein.The median white blood cell count was 12.68×10 9 /L, with lymphocytes predominating (median lymphocyte percentage: 65.70%), consistent with a typical response to viral infection.
Platelets: The median platelet count was 211×10 9 /L, within the normal range but leaning towards the lower limit, suggesting a potential trend towards mild thrombocytopenia in some patients.
Liver Function Tests: Median levels of ALT (47.5 U/L) and AST (51.2 U/L) were elevated, corroborating the observed hepatomegaly and indicating EBV's impact on hepatic function.
Biochemical Panel: The panel also measured total bilirubin (TBiL) and lactate dehydrogenase (LDH), which are critical for assessing liver damage and cellular turnover, respectively.
The baseline characteristics and clinical manifestations elucidated in this study lay the groundwork for understanding the specific impacts of EBV in pediatric populations.Notable trends, such as the high prevalence of fever and organomegaly, are vital for effective diagnosis and management of EBV infections.Moreover, the comprehensive analysis of hematological and immunological parameters offers a detailed snapshot of the infection's clinical profile, enabling the development of targeted therapeutic strategies.

| Binary classification of EBV-DNA copy numbers in plasma and correlation with clinical indicators
This study further analyzed the association between EBV-DNA copy numbers in plasma and various clinical indicators using a binary classification approach.The cohort was divided based on the median EBV-DNA copy numbers in plasma into two groups: high EBV-DNA copy numbers in plasma (>38.9 × 10 2 copies/ml) and low EBV-DNA copy numbers in plasma (≤38.9 × 10 2 copies/ml).This classification facilitated a detailed comparison of clinical and laboratory characteristics between the groups.

| Clinical and laboratory findings
Fever Duration and Intensity: There were significant differences in the duration of fever between the groups, with the high EBV-DNA copy numbers in plasma group experiencing longer fever durations (p = .0022),suggestive of a more severe or protracted infection phase.
Hematological Indicators: The platelet count was notably lower in the high EBV-DNA copy numbers in plasma group (p = .0212),indicating a possible aggravation of thrombocytopenic conditions in patients with higher EBV loads.
Liver Function: ALT levels were significantly elevated in the high EBV-DNA copy numbers in plasma group (p = .001),aligning with increased hepatic involvement or damage associated with higher viral loads.
Immunological Response: Significant differences were observed in immunoglobulin levels (IgM, p = .0039;IgG, p = .0195),pointing towards a heightened immune response or altered B-cell dynamics in patients with higher EBV-DNA copy numbers in plasma.
The binary classification revealed critical variances in clinical outcomes related to EBV-DNA copy numbers in plasma.Notable correlations included: Inflammatory Markers: Increased lactate dehydrogenase (LDH) levels in the high EBV-DNA copy numbers in plasma group (p = 0.0001) were indicative of higher cell turnover or tissue damage.Bilirubin Levels: Total bilirubin (TBiL) was also significantly higher in patients with elevated EBV-DNA copy numbers in plasma (p = .025),suggesting enhanced bilirubin metabolism or liver dysfunction associated with active infection.
Table 2 displays the findings that reveal notable variations in various clinical parameters between the two groups of children infected with EBV, These findings underscore the potential of EBV-DNA copy numbers in plasma as a biomarker for assessing the severity of infection and guiding clinical management.The differences in clinical and immunological indicators between the two groups highlight the impact of viral load on disease manifestations and severity.The association of higher EBV-DNA copy numbers in plasma with more severe clinical features and laboratory abnormalities provides evidence for predicting patient outcomes and tailoring therapeutic interventions.

| Binary classification of hospital days and correlation with clinical indicators
In this analysis, the study cohort was further divided based on the median hospital stay into two groups: longer hospital stays (>9 days) and shorter hospital stays (≤9 days).This stratification allowed for an examination of the association between the length of hospitalization and various clinical and laboratory parameters, potentially indicative of more severe disease states or complications.

| Key clinical and laboratory variations
Tonsil Enlargement and Inflammation: Significant differences were observed in tonsil enlargement (p = .0024),with the longer stay group showing a higher prevalence, suggesting more severe oropharyngeal involvement.
Hematological Parameters: A lower platelet count was noted in the group with longer hospital stays (p = .0045),which may be reflective of prolonged disease activity or secondary complications affecting bone marrow function.

| Immune response and organ function
LDH Levels: Elevated LDH levels in the longer stay group (p = .0394)indicated increased cellular turnover, which could be associated with more extensive tissue damage or a higher degree of inflammatory response.

| Statistical insights and clinical implications
Binary Classification Analysis: The findings from the binary classification according to hospital stay duration revealed noteworthy differences in several key clinical indicators, directly correlating with the intensity and progression of EBV-associated symptoms and complications.
Inflammatory and Immune Markers: Ferritin levels, an acute phase reactant, were significantly higher in the longer stay group (p = .0106),reflecting a more intense inflammatory response or ongoing infection.
EBV-DNA copy numbers in plasma: Furthermore, a significant difference in EBV-DNA copy numbers in plasma (p = 0.0361) between the groups highlights the potential of viral load as a predictor of disease severity and the necessity for prolonged hospitalization.
Table 3 displays notable variances in the duration of hospital stay in pediatric EBV infections is closely linked with several clinical and laboratory parameters, indicating its utility in assessing disease severity.The associations observed support the use of these parameters in clinical practice to identify patients at risk of severe outcomes, thereby facilitating timely and targeted interventions.

| DISCUSSION
Similar to other herpesviruses, EBV invades epithelial cells and circulating B lymphocytes following initial infection, remaining latent in the body for prolonged periods. 11If the balance between the host and the virus is disturbed, the virus may reactivate.Various diseases are linked to primary or reactive EBV infections, including infectious mononucleosis (IM), respiratory infections, encephalitis, malignant lymphomas, nasopharyngeal carcinoma, aplastic anemia, hemophagocytic lymphohistiocytosis (HLH), immunodeficiency, and autoimmune diseases. 12iagnosing EBV infection primarily depends on serological tests and molecular biology methods.However, the accuracy of serological tests may be compromised by the underdeveloped immune system in children and the presence of maternal antibodies in infants, leading to false-negative or false-positive results. 13olecular biology testing has become an essential tool for diagnosing and monitoring EBV infections and associated diseases in immunocompromised hosts. 14rior research indicates 15 that serological and molecular biology tests should be used complementarily to diagnose a patient's EBV infection status. 16n our study, We grouped these patients based on EBV copy numbers and hospital stay durations, compared clinical indicators across groups, and clarified the T A B L E 2 Binary Classification Analysis of EBV-DNA copy numbers in plasma and Clinical Indicators.

Characteristic Variable
EBV-DNA copy numbers in plasma (×10 2 ) >38.9* EBV-DNA copy numbers in plasma (×10 2 ) ≤38.9This suggests that EBV-DNA copy numbers in plasma might correlate with these clinical variables.Additionally, the classification based on hospital days revealed significant differences in tonsil enlargement, platelet count, LDH, ferritin, and EBV-DNA copy numbers in plasma, suggesting a potential correlation between hospital stay duration and these clinical variables.
Children in the high copy number group also had higher ALT and TBiL levels, indicating that higher EBV-DNA copy numbers in plasma are more likely to cause liver damage, consistent with findings by Lin Shengjing. 17Therefore, children in the high copy number group require more active monitoring of liver function.Fever is the most common clinical manifestation in children with EBV infections, and the duration of fever often varies.Some children with EBV infections need hospital treatment due to their condition, and due to individual differences in disease progression, the duration of hospital stays also varies.Our study found that children in the high copy number group had longer fever and hospital stay durations.Thus, EBV-DNA copy numbers in plasma can be an important indicator for assessing the duration of fever and clinical outcomes in children.However, this indicator is limited by factors such as the inability to perform and long turnaround times for results in some primary care hospitals.
Our findings also showed that platelet count and LDH correlate with EBV-DNA copy numbers in plasma, suggesting that these indicators can be used to assess the duration of fever and clinical outcomes in children.To further validate this observation, our study classified hospital stay durations based on the median value, and the analysis yielded similar results.Moreover, these indicators are more readily available and have higher clinical testing efficiency in primary care hospitals, thus holding greater clinical value.
Our findings demonstrate that EBV viral load and the subsequent length of hospital stay are correlated with clinical indicators.Platelets, small anucleate cells circulating in the blood for about 7 to 10 days, primarily function in hemostasis by forming clots to protect the integrity of blood vessels.They originate from megakaryocytes, which are large polyploid cells found in the bone marrow and are developed from hematopoietic stem cells.As megakaryocytes mature internally, they develop proplatelets that release into the bloodstream. 18Platelets typically circulate at levels between 150 and 450×10 9 /L when bone marrow is functioning normally. 19Different cytokines, such as stromal cell-derived factor 1 (SDF-1), granulocytemacrophage colony-stimulating factor (GM-CSF), interleukins (IL-3, IL-6, and IL-11), fibroblast growth factor 4 (FGF-4), and thrombopoietin (TPO), play a role in stimulating the production of megakaryocytes, with TPO being the most important. 20While TPO plays a vital role in supporting hematopoietic stem cells, 21 the majority of these cytokines have pro-inflammatory properties and can prompt the quick maturation and activation of white blood cells, leading to an increase in megakaryocyte production.This paper elucidates the impact of inflammatory processes on platelet production.
Previous research suggested that acute EBV infections could lead to thrombocytopenia in children. 22A recent study by Zhang et al. 23 Reported a rare case involving a 14-year-old girl experiencing severe thrombocytopenia, with a platelet count dropping to 5×10 9 /L, and showing signs of spontaneous bleeding along with periorbital edema, which is an uncommon symptom of EBV-related infectious mononucleosis.After receiving intravenous immunoglobulin and a 4-day regimen of methylprednisolone, leading to her discharge 7 days after being admitted a platelet count of 143×10 9 /L.It is important to keep in mind the possibility of EBV infection when diagnosing acute severe thrombocytopenia.
Lactate accumulation typically results in a lactate (LA) microenvironment, often associated with worse outcomes in cancer progression, metastasis, and decreased diseasefree and overall survival. 24Lactate dehydrogenase (LDH) is found in many different tissues and can be detected in the blood because it helps convert pyruvate to lactate, a reaction that is rapid and near-equilibrium, highly dependent on local lactate gradients. 25An increase in LDH and lactate production is considered a poor prognostic aspect in several malignancies including solid tumors, 26 but the mechanisms by which oncogenic viruses exploit this metabolic process for cancer progression and adaptation to acidic conditions are still not well understood.Evidence indicates that dynamic interactions between EBV and surrounding environmental factors, such as hypoxic stress, is closely linked to the aggressive behavior of host cells.
EBV has the ability to stimulate anaerobic glycolysis in NPC and B-cell lymphomas by upregulating the oncogenic proteins LMP1 and HIF-1α. 27Our findings support that LDH could be a significant marker reflecting EBV viral effects.
LDH is an enzyme widely present in various tissue cells of the human body.When cells are damaged or undergo necrosis, LDH is released from within the cells into the bloodstream, reflecting the metabolic state and the extent of tissue damage. 28As an intracellular enzyme, LDH is released into the blood when cells are damaged or necrotic, leading to elevated serum LDH levels, which indicate the degree of cellular necrosis and the immune status of the body.In this study, we grouped these patients based on EBV copy numbers and hospitalization duration.The results showed statistically significant differences in LDH levels among the groups, indicating that higher plasma EBV DNA copy numbers are more likely to induce immune-inflammatory responses and inflammatory damage, resulting in prolonged hospitalization in affected children.
This study is primarily limited by its retrospective design and dependence on data from a single center, which may contribute to selection bias and limited regional representativeness.Future studies might consider more detailed subgroup analyses based on age, sex, and severity of clinical manifestations to determine these factors' specific effects on the clinical progression of EBV infections.A prospective cohort design and expansion to multiple centers would help reduce information bias in retrospective studies, enhance the generalizability of the research, and improve the reliability of the conclusions.Additionally, controlling for more potential confounders and increasing the sample size are recommended.

| CONCLUSION
Infectious mononucleosis, caused by EBV infection, is a common disease in children with varying degrees of severity.Some children require hospitalization, and those with longer hospital stays have higher LDH levels compared to those with shorter stays.Our study confirms that changes in platelet counts and lactate dehydrogenase levels may serve as important indicators of clinical outcomes in children with EBV infections, providing new monitoring markers for clinical treatment.
Basic information of children.
T A B L E 1 Ferritin (ug/L) 110.3 (73.1-163.9)(Continues) Binary Classification of Hospital Days and Correlation with Clinical Indicators.
T A B L E 3 *Optimal cut-off value: 9.